The SNCA gene is found on chromosome 4, position 4q22.1; it is organized into 10 exons ²⁰ -the size ranges between 42 and 110 base pairs ²¹- and spans about 117 kb. ²⁰

SNCA is an abundant protein that represents 1% of proteins soluble in cytosol in the brain. ²¹ It is expressed mainly in the pre-synaptic nerve terminals of the brain and in low concentrations in all other tissues, except the liver. ²⁰ This protein is found in at least 4 different alternative splicing variants for functional polypeptide synthesis. ¹⁷ The predominant variant in humans is composed of140 amino acid residues whose mRNA is synthesized from the transcription and processing of 6 exons ²⁰; the second isoform is 126 amino acids long and is produced by the exclusion of exon 3; the third isoform is 112 amino acids long product of the exclusion of exon 5 ^20,21; and the fourth isoform is 98 amino acids long with deletion of exons 3 and 5 ^21,17 (Figure 2).

Figure 2 Source: Own elaboration based on Janeczek et al.¹⁷.

SNCA has been particularly linked with synapse and plasticity processes. The high concentration in presynaptic terminals involves the protein in the recycling and maintenance of the synaptic vesicles ²¹; mediation functions have also been attributed to dopaminergic neurotransmission ^20,22,23, including synthesis, storage, release and reabsorption of the neurotransmitter. Although the functions of SNCA have not been fully elucidated, the most relevant have been related to neuroprotection:

Suppression of dopaminergic neuron apoptosis by deactivation of the nuclear factor Kb (NF-kB) and reduction of protein kinase C (PKC) transcription and activity. ²⁴

On the other hand, a pathophysiological model of the possible mechanisms in which SNCA aggregation is toxic to neurons has been proposed, and is related to post-translational modifications such as phosphorylation and oxidation that influence the binding to membrane lipids; SNCA monomers form dimers that may or may not propagate. The latter generate oligomers that interact with the cytoplasmic membrane forming pores and inducing intracellular calcium flow abnormally. ²⁸

Amyloid fibrils formation generates accumulation of intracellular inclusions, also known as Lewy bodies, which, due to their toxicity, block the trafficking of proteins from the endoplasmic reticulum up to the Golgi apparatus and alter mitochondrial function, protein degradation and synaptic transmission, causing accumulation in the cytoplasm. ^28,29

These phenomena increase membrane conductance, activate microglia and result in dysfunctional synapses, leading to disorders known collectively as α-synucleopathies ^30,31, which include Parkinson's disease, Lewy body dementia and multisystem atrophy; it is also found secondarily in Alzheimer's disease. ²⁹ The passive release of SNCA from damaged neurons could be relevant for neurodegenerative processes, since it is known that neuron-to-neuron propagation is an important phenomenon in the progression of these pathologies and affects different brain areas and structures. ^32-34

In this context, SNCA is observed in both physiological and pathological conditions. In the first case, it has multiple functions related to cellular regulation, while in the second, the modifications caused by inflammatory processes or toxicity can contribute to the abnormal folding of the protein, leading to important pathological consequences. Research in this regard has been directed to neurodegenerative diseases and addictions, especially alcohol dependence, because SNCA alteration affects surface expression and dopamine uptake, altering the reward pathway associated with addiction during chronic alcohol consumption. ^17,35

The flow of SNCA between the brain and peripheral tissues could have important pathophysiological implications. When the protein is radioactively labeled, passage through the blood-brain barrier in the brain-blood direction and vice versa is observed; this is the mechanism responsible for regulating the brain level, therefore, accumulation of SNCA in the brain is observed in cases of inflammatory processes that lead to increased neurotoxicity due to impaired blood flow. ₍₁₆₎ It has been shown that the injection of a single dose of lipopolysaccharide (LPS) to induce increased vascular permeability increases SNCA expression, which is associated with increased opening of the blood-brain barrier. ¹⁸

Total SNCA concentrations in blood cells and saliva are determined by ELISA test adapted for maximum detection. ^15,36 In turn, it has been evaluated in different tissues, cells and human fluids ^15,37 such as blood ^38,39, erythrocytes ^15,40, saliva ⁴¹ and cerebrospinal fluid (CSF). On the other hand, erythrocyte expression is regulated by post-translational mechanisms through SNCA degradation and release, and is associated with the plasma membrane of erythrocytes, being more prone for aggregation and easier for detection there than in neuronal cells. ^41-43

With all of this in mind, the detection of extracellular SNCA represents a reliable marker ⁴⁴, since the neuronal protein is secreted in the cerebral environment circulating to the cerebrospinal fluid (CSF) and then to the blood. ³⁸ In addition, studies have been conducted in alcoholic subjects and have concluded that the elevation of mRNA levels is common in humans, rodents and primates. Also, the protein has been detected in plasma and its increase has been directly related to the increase in mRNA. ⁴⁵ Consequently, SNCA could be useful as a peripheral marker of alcoholism, although more studies are required. ^8,10,11

SNCA gene expression has been studied in different models. In mice, results have been contradictory, since possible changes in expression were evaluated after short periods of abstinence; basically, mice had constant access to an 8% alcohol solution for 32 days, then techniques were used to quantify the gene and protein levels in brain and blood. In the brain, the results showed no changes in mRNA in any of the areas studied, but the protein level was modified by an increase in the amygdala after 24 hours of abstinence, which did not correspond to the level of mRNA transcription; the authors attributed this to a possible accumulation in synaptic boutons due to alterations in transport or degradation. On the contrary, an increase in gene expression was observed in blood at 48 hours of abstinence, which in this case was not related to brain expression. ⁸

An important reduction of SNCA mRNA expression has been evidenced in the prefrontal cortex of human alcoholics with the most common variant of SNCA-140. ^6,7,9 In contrast, in animal models, SNCA expression increased in regions of the brain such as the hippocampus and the caudate, and decreased in the region of the striatum. ⁹

Furthermore, in non-human primates with self-administration of alcohol for 18 months and with a diagnosis of alcoholism, a 3.21-fold increase in the levels of the SNCA gene in peripheral blood was observed. ¹⁰ In humans, mRNA expression and proteins in whole blood were evaluated in subjects with a withdrawal period of 24-72 hours, showing in both cases a significant increase in the expression of SNCA compared to control subjects. ^11,44

A polymorphism is an alternative form of an allele with a frequency >1% in a given population. ⁴⁶ There are several types of polymorphisms, but the most frequent are simple nucleotide (SNP) polymorphisms; most of them have two alleles that are represented by the substitution of one base for another. ⁴⁶ SNPs are characterized by stability, simplicity and high frequency and are genetic markers used to determine the risk of presenting a pathology and the response to pharmacological treatment. ³⁵

The identification of polymorphisms in the promoter region of the SNCA gene is of particular interest in Parkinson's and Alzheimer's diseases. Research has been conducted in several countries in which different polymorphisms associated with these diseases have been identified and reported. ^9,22,47

The SNCA gene has been studied because it is highly polymorphic. SNCA-Rep 1 is a polymorphic microsatellite that is upstream of the gene and is associated with use, abuse, dependence and compulsive craving for alcohol phenotypes. ¹² Findings in human blood have revealed an association between the long alleles of the SNCA-Rep 1 polymorphism (271pb) and increased expression in dependent patients. On the other hand, there was a greater frequency of the short allele of SNCA-Rep 1 (267pb) in human brain tissue of the prefrontal cortex, which was associated with decreased expression of SNCA; this suggested that individuals with at least one copy of the 267-bp allele are more susceptible to presenting an alcohol dependence and abuse phenotype. ⁷

The role of SNCA in the dependence and compulsive craving phenotypes was evaluated in 219 American families of European descent. It was found that there was no association between these polymorphisms and alcohol dependence; however, for the phenotype of compulsive craving for alcohol, it was possible to identify five polymorphisms in the gene promoter region (upstream): rs7678651, rs7687945, rs2736995, rs2619364 and rs2301134, of which two were within the eight SNPs previously associated with this phenotype (rs2736995, rs2619364, rs2583985, rs2737006, rs3561184, rs356183, rs356195 andrs3561168)¹⁹(Figure 3). This finding is of relevance taking into account that polymorphisms in the promoter region can influence the transcription of the gene and therefore its expression.

Figure 3 Source: Own elaboration based on Foroud ef al.¹⁹.

The regulation of gene expression is affected by independent changes in the nucleotide DNA sequencing, object of study of epigenetics. In vertebrates, several phenomena of epigenetic nature occur, including methylation, perhaps the most studied. It consists of the enzymatic addition of a methyl group on carbon 5 of the cytosine; a large part of the 5-methylcytosines (5mC) is found in the CpG islands and in the complementary chain of the 3'-GpC-5 'dinucleotide. These islands are located between the central region of the promoter and the transcription start site, for which a decrease in the expression is observed when they are hypermethylated. ⁴⁸ Methylation in cytosine residues causes a variation in the structure of chromatin in terms of spatial disposition and temporality, with an inverse correlation between DNA methylation levels and gene expression. DNA methylation is an epigenetic marker involved in gene regulation and is vital for maintaining normal gene silencing, genomic imprinting and inactivation of the X chromosome. ⁴⁸

The overall DNA methylation of peripheral blood mononuclear cells and, in particular, the promoter region of the SNCA gene, is altered by changes in plasma homocysteine concentrations in disorders associated with alcohol consumption. ⁴⁹ Moreover, the physical interaction of SNCA with DNA-methy !transferase-1 in the nucleus seems to decrease DNA methylation by increasing the enzyme in the cytosol due to retention; this results in a significant increase in expression levels. ⁵⁰

Another study with 84 chronic alcoholics and 93 controls investigated whether DNA methylation in the promoter region of the SNCA was altered in peripheral blood mononuclear cells, both in patients intoxicated with alcohol and with early alcohol abstinence. Hypermethylation was observed in the promoter region of SNCA, which was associated with increased levels of homocysteine. ^49,51

The influence of CpG islands methylation on the expression of the SNCA gene does not seem to be limited to the promoter region of the gene that regulates transcription, but extends to other regions such as intron 1 and exon 1 ^52-54, which have been studied in α-synucleopathies; nevertheless, to date, there are no studies oriented to alcoholism. Due to the limited evidence on the changes of SNCA methylation in alcoholics, it is necessary to do more research to know the influence of methylation on mRNA expression.

MiRNAs, another regulatory mechanism, are non-coding oligonucleotides that have an important role in the post-transcriptional regulation of gene expression and mRNA degradation. They are widely found in the brain, affecting its development, neuronal differentiation, synapses and neurogenesis. ⁵⁵ MiRNAs intervene in adaptations after exposure to alcohol in cellular, animal and human models ¹⁶; in alcoholics, a relationship between reduced gene expression and the ]presence of around 35 different types of miRNA has been identified. ⁵⁵

MicroRNA-7 expressed in neurons represses α-synuclein expression through the three prime untranslated region (3'-UTR) of the mRNA, and inhibits cell death mediated by SNCA; however, in mice with dopaminergic neurotoxicity with MPTP injections (neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and in cells that were transfected with constructs that expressed α-synuclein, in the absence or presence of miRNA-7 and exposure to hydrogen peroxide, it was observed that the decrease of the expression of miR-7 increases the expression of SNCA. ⁵⁵

This is confirmed by recent studies in mice injected with a lentiviral vector that mediates miRNA-7 expression in complementary binding sites to induce the loss of miRNA-7 function, where there was an increase in SNCA expression in vitro and in vivo, dopaminergic neuronal death and reduction of striatal dopamine. ¹⁴

After exposure of cerebral cortical neural precursors of mouse fetus to doses of alcohol equivalent to those consumed by alcoholics, suppression was observed in the expression of four miRNA: miR-21, 335, 9 and 153. ⁵⁶ MiRNA-153 acts to degrade mRNA and has been identified as a regulator of the levels of SNCA expression, which, when overexpressed together with miRNA-7, significantly reduces the expression of SCNA and its reduction increases it. ¹³

In short, as a result of genetic or epigenetic modifications in SNCA, the polymorphisms, the methylation status of CpG islands and miRNAs contribute to the differential expression of SNCA and to neuroadaptation (Figure 4).

Figure 4 * They seem to influence SNCA expression, but there is still no research in subjects with high alcohol consumption. Source: Own elaboration.